1. Technical Field
The subject invention relates to improvements in nucleic acid isolation, and more particularly, relates to modifications to the subtractive hybridization method and to reagents such as oligonucleotides that are useful when performing the method.
2. Background Information
Changes to the presence, level, or sequence of a particular nucleic acid can have a significant effect on the host in which the nucleic acid resides. The identification and isolation of such nucleic acid sequences are essential to their analysis and understanding. To this end, the approaches of genetics, infectivity studies, comparative nucleic acid fingerprinting, and subtractive hybridization have been used.
Subtractive hybridization methods enrich for nucleic acid sequences present in one sample but absent, decreased, or altered in an otherwise identical sample. For a review, see O. D. Ermolaeva et al., Genetic Anal.: Biomol. Eng. 13:49-58 (1996). A xe2x80x9ctargetxe2x80x9d in such methods is the set of nucleic acid sequences to be enriched, and the xe2x80x9ctester and driverxe2x80x9d are nearly identical nucleic acid samples that preferably differ from one another only by the presence or absence of the target sequence(s) respectively.
Generally, in subtractive hybridization, driver and tester nucleic acid are extracted from the samples; cDNA then is prepared if the nucleic acid of interest is RNA; driver DNA and tester DNA are fragmented and one or the other is modified to enable subsequent purification; and finally, a mixture of the fragmented DNAs, in which driver is in substantial excess over tester, is heat denatured and complementary single strands are allowed to reanneal. Due to the excess of driver versus tester, a majority of tester sequences held in common with driver will exist as tester/driver hybrids. Species containing sequences common to driver and tester are eliminated by means of the described modification, leaving a tester-only population enriched in target sequences. If further enrichment is required, additional rounds of subtraction are performed. Finally, individual fragments cloned from the subtraction products are screened for target sequences (i.e., those sequences present in tester but absent, or significantly reduced, in driver) (O. D. Ermolaeva et al., supra).
Representational Difference Analysis (RDA), like other recent methods of subtractive hybridization incorporate the polymerase chain reaction (PCR) as an integral part of the procedure (U.S. Pat. No. 4,683,195; Saiki et al., Science 230:1350-1354 (1985)). The success of PCR-based subtractive hybridization is partially dependent on the initial amplicon complexity and/or the relative abundance of target sequence within the amplicon. (An amplicon may be defined as the entity comprising the set of nucleic acid sequences amplified by PCR.) If the complexity is too high, or if the target sequence concentration is too low, the kinetics of hybridization prevent effective enrichment, and the method fails.
Amplicon complexity is reduced in the RDA procedure by the amplification of only a representative subset of all possible fragments from driver and tester. Such subsets are achieved by selective amplification of nucleic acid fragments based on size. Alternatively, the starting nucleic acid can be enriched for target sequences prior to subtraction by partial purification, accomplished by passing the sample through a two-micron filter prior to extraction, thereby eliminating most of the cellular nucleic acids present in the sample and alleviating the necessity of reducing amplicon complexity (Simons et al., Proc. Natl. Aca. Sci. USA 92:3401-3405 (1995)).
Other factors also are likely to affect performance in this method. For example, differences in reassociation rates and PCR efficiencies between fragments impose a strong selection for those products most readily formed, regardless of whether or not the sequence is unique to tester. If such a sequence is not unique, it could overwhelm the subtractive capacity of the driver especially in later rounds, resulting in the isolation of sequences that are not specific to, or elevated in, tester-versus-driver. Regardless of their source, such xe2x80x9cfavoredxe2x80x9d sequences tend to dominate the enriched fragment population and out-compete tester-unique products that are less efficiently formed, making tester-unique product detection difficult. This problem recently has been approached by isolating the major enrichment products obtained after a series of subtractions and adding them back individually to the driver, thereby boosting the subtractive capacity for those sequences (Ushijima et al., Proc. Natl. Acad. Sci. USA 94: 2284-2289 (1997) and Hubank et al., Nucleic Acids Res. 22:5640-5648 (1994)). However, a second series of subtractions then must be performed to isolate tester-unique sequences not previously obtained, such as lower copy number sequences or those that amplify relatively poorly.
Problems associated with high amplicon complexity and low copy numbers in the tester have not been fully addressed or resolved. These factors can negatively affect the isolation of sequences by reducing the sensitivity of the subtraction procedure.
In view of the above discussion, it certainly would be advantageous to provide modifications to the subtractive hybridization procedure, including RDA, which would address the problems associated with high amplicon complexity and low copy numbers in the tester.
All U.S. patents and publications are hereby incorporated in their entirety by reference.
The present invention provides a modified subtractive hybridization method termed Selectively Primed Adaptive Driver-RDA (xe2x80x9cSPAD-RDAxe2x80x9d), which utilizes a driver-versus-driver subtraction control performed in parallel with the driver-versus-tester subtraction step. The products of the driver control subtraction from each round can be used as the driver of the subsequent round.
This method for performing subtractive hybridization uses a tester sample and a driver sample to determine the presence of a nucleic acid sequence difference in the tester sample. In detail, the method comprises the steps of: (a) separately isolating total nucleic acid from the tester sample and the driver sample, and generating double-stranded cDNA/DNA from the total nucleic acid from the tester sample and the driver sample; (b) digesting the double-stranded cDNA/DNA generated from the tester sample and the driver sample of step (a) with a restriction endonuclease in order to produce a set of restriction fragments for each sample; (c) ligating the driver and tester restriction fragments of each set of step (b) to an oligonucleotide adapter set 1, and amplifying the resulting products with selective primers such that a subset of the restriction fragments of step (b) is amplified;(d) removing the selective primers sequences by restriction endonuclease digestion in order to produce tester and driver amplicons, ligating the 5xe2x80x2-ends of said driver and tester amplicons to an oligonucleotide adapter set 2 to form driver-control and tester, mixing driver-control and tester separately with an excess of non-ligated driver amplicon each, denaturing the resulting mixtures, and allowing the denatured nucleic acid strands within each mixture to hybridize; (e) filling in the 3xe2x80x2-ends of the reannealed driver/tester and the reannealed driver/driver-control using a thermostable DNA polymerase and amplifying resulting sequences; (f) removing remaining single-stranded DNA by digesting with a single-stranded DNA nuclease and amplifying; (g) amplifying double-stranded DNA remaining after nuclease digestion; and (h) cleaving subtraction products of the driver/tester and driver/driver-control with a restriction endonuclease to remove oligonucleotide adapters, and repeating steps (c) through (h), wherein steps (c) through (h) utilize an oligonucleotide adapter set not used in any previous round of RDA, wherein one round consists of performance of RDA steps (c) through (h), and utilize as driver, for each new round of RDA, the restriction endonuclease-cleaved product of the driver/driver-control subtraction from immediately preceeding steps (c) through (h). (Steps (c) through (h) can be repeated for any desired number of times.) In this method, the restriction endonuclease of step (b) and/or step (h) may be a 4-6 basepair recognition site (with an overhanging 5xe2x80x2 end, preferably a 4 basepair recognition site. The restriction endonuclease may be, for example, Sau3AI. Although less preferred, any restriction enzyme with a palindromic tetra- or hexanucleotide recognition sequence may be used. The choice of enzyme will affect both amplicon complexity and the design of the oligonucleotide adaptors. Amplicon complexity is increased when a restriction endonuclease with a 4 bp recognition site is used, relative to the complexity obtained when a restriction endonuclease with a 6 bp recognition site is used. This is due to the greater number of amplifiable sequences in the former vs. the latter. Furthermore, the choice of restriction endonuclease affects oligonucleotide adaptor design since the adaptor must be compatible with the sequence and structure present on the ends of the restriction endonuclease digested DNA in order for efficient ligation to occur.
The present invention also includes a method for visual identification of unique tester sequences comprising the steps of: (a) separately isolating total nucleic acid from a tester sample and a driver sample, and generating double-stranded cDNA/DNA from the total nucleic acid from the tester sample and the driver sample; (b) digesting the double-stranded cDNA/DNA generated from the tester sample and the driver sample of step (a) with a restriction endonuclease in order to produce a set of restriction fragments for each sample; (c) ligating the driver and tester restriction fragments of step (b) to an oligonucleotide adapter set 1, and amplifying the resulting products with selective primers such that a subset of the restriction fragments of step (b) is amplified; (d) removing the selective primers sequences by restriction endonuclease digestion in order to produce tester and driver amplicons, ligating the 5xe2x80x2-ends of the driver and tester amplicons to an oligonucleotide adapter set 2 to form driver-control and tester, mixing driver-control and tester separately with an excess of non-ligated driver amplicon each, denaturing said resulting mixtures, and allowing the denatured nucleic acid strands within each mixture to hybridize; (e) filling in the 3xe2x80x2-ends of the reannealed driver/tester and the reannealed driver/driver-control using a thermostable DNA polymerase and amplifying resulting sequences; (f) removing remaining single-stranded DNA by digesting with a single-stranded DNA nuclease and amplifying; (g) placing the driver-tester and driver-control products on a solid substrate; and (h) visually identifying driver tester bands not present in the driver-control bands.